Thiopurine S-Methyltransferase (TPMT) catalyzes thiopurine S-methylation, an important metabolic pathway for drugs such as 6-mercaptopurine (6-MP). 6-MP acts as a thiopurine antimetabolite, and is commonly used in the therapy of acute lymphoblastic leukemia (ALL), ulcerative colitis and Chron's disease, inflammatory bowel disease, and other diseases. 6-MP can be acted upon by several metabolic routes, one of which generates 6-Thioguanine nucleotides (6-TGN), the antimetabolite responsible for 6-MP's therapeutic effects. An alternative metabolic route involves the S-methylation of 6-MP, which leads to its inactivation. This led to studies of a correlation between TPMT activity levels and response to 6-MP therapy (see Lennard et al., Lancet, 336:225-229, 1990, herein incorporated by reference). The results of this study showed that children with lower 6-TGN concentrations had higher TPMT activities and a higher subsequent relapse rate, indicating that genetically determined TPMT activity may be an important regulator of the cytotoxic effect of 6-MP.
The most common allele for TPMT deficiency is designated TMPT*3A, which consists of 2 SNPs, G460A and A719G, both of which lead to amino acid changes in the TPMT enzyme. While both mutations together cause a complete loss of activity of the TPMT enzyme, the G460A mutation, when alone, causes a 9-fold reduction in TPMT enzymatic activity. The G460A mutation, in the absence of the second polymorphism A719G, is designated TPMT*3B.
While the TPMT gene is located on chromosome 6p22.3, a highly homologous TPMT pseudogene is located on chromosome 18. Due to the high sequence similarity of the two genes, detection assays designed to interrogate base 460 may be compromised due to interference caused by the presence of the pseudogene. This type of interference may be present when attempting to interrogate other polymorphic sequences present in the genome of a given organism with detection assays. As such, what is needed are methods and compositions for use with detection assays, particularly hybridization based detection assay that minimize or eliminate the interference that can be caused by interfering sequences.